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Measuring cristae and inner boundary membrane potential gradient a Representative images of HeLa cells expressing mt-sfGFP (green), stained with 81, 40.5, 13.5, 5.4, 2.7, or 1.35 nM tetramethylrhodamine methyl ester (TMRM) (magenta) and examined using simultaneous dual-color 3D-SIM. b Intensity line plots of the mitochondrion in the white box in a showing the mt-sfGFP and TMRM intensity distribution. Full width at half maximum (FWHM) of mt-sfGFP and TMRM distributions are indicated with the corresponding maximal (Imax) and half-maximal (Imax/2) intensities. c Schematic illustration of the calculation of the IBM association index on the example of HeLa cells expressing mt-sfGFP (green) and stained with 81 nM TMRM (magenta). The reference channel (mt-sfGFP) is thresholded and split by erosion and dilation into inner boundary membrane (IBM) and cristae membrane (CM) related segments, which are used as masks to measure TMRM mean intensities. The ratio of IBM intensity (IIBM,TMRM) and CM intensity (ICM,TMRM) results in the IBM association index. d Quantitative analysis of a using the ∆FWHM of TMRM and mt-sfGFP (described in b) or the IBM association index (described in c) to determine TMRM distribution to the IBM. The higher ∆FWHM or IBM association index, the broader the TMRM distribution indicating a stronger TMRM staining in the IBM. Data information: Horizontal lines in d represent the median, the lower and upper hinge show, respectively, first quartile and third quartile, and lower and upper whiskers encompass minimal and maximal values. Images and analyses were obtained from each 9–10 cells in 6 independent experimental days (n = 6). *P < 0.05 vs. 13.5 nM, #P < 0.05 vs. 40.5 nM and ⁺P < 0.05 vs. 81 nM TMRM conditions evaluated using one-way analysis of variance (ANOVA) with Bonferroni post hoc test.
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Mitochondrial ultrastructure represents a pinnacle of form and function, with the inner mitochondrial membrane (IMM) forming isolated pockets of cristae membrane (CM), separated from the inner-boundary membrane (IBM) by cristae junctions (CJ). Applying structured illumination and electron microscopy, a novel and fundamental function of MICU1 in med...
Citations
... For that purpose, we developed a method that builds on fluorescence intensity distribution in a model limited to mitochondrial geometry to analyze the CM/IBM membrane potential distribution in living cells. We used IMM selective and specific dyes MitoTracker™ Green FM (MTG) and tetramethylrhodamine methyl ester (TMRM) 7,8,11,13 . Without the requirement of the transfection of genetically encoded sensors, this method allows us to track changes in membrane potential gradients over time and to correlate them with mitochondrial ATP-production and morphology. ...
... Notably, the distribution of TMRM depended on the TMRM affinity to the higher (more negative) membrane potential and the concentration-dependent saturation of the dye in mitochondria at high (81-40.5 nM) concentrations 7 . Low concentrations (2.7-1.35 ...
... We employed two distinct analysis methods for this purpose. (I) The IBM association index was previously used to determine the translocation of proteins between the cristae and IBM 4,7 . This index is fully automated. ...
The complex architecture and biochemistry of the inner mitochondrial membrane generate ultra-structures with different phospholipid and protein compositions, shapes, characteristics, and functions. The crista junction (CJ) serves as an important barrier separating the cristae (CM) and inner boundary membranes (IBM). Thereby CJ regulates the movement of ions and ensures distinct electrical potentials across the cristae (ΔΨC) and inner boundary (ΔΨIBM) membranes. We have developed a robust and flexible approach to visualize the CJ permeability with super-resolution microscopy as a readout of local mitochondrial membrane potential (ΔΨmito) fluctuations. This method involves analyzing the distribution of TMRM fluorescence intensity in a model that is restricted to the mitochondrial geometry. We show that mitochondrial Ca²⁺ elevation hyperpolarizes the CM most likely caused by Ca²⁺ sensitive increase of mitochondrial tricarboxylic acid cycle (TCA) and subsequent oxidative phosphorylation (OXPHOS) activity in the cristae. Dynamic multi-parameter correlation measurements of spatial mitochondrial membrane potential gradients, ATP levels, and mitochondrial morphometrics revealed a CJ-based membrane potential overflow valve mechanism protecting the mitochondrial integrity during excessive cristae hyperpolarization.
... 52,53 Mammalian MICU1 is also considered a 'plug' regulating calcium fluxes upon methylation by PRMT1 during aging or cancer. 54 Less is known about the molecular cues controlling organellar calcium levels relevant to the overall homeostasis of this cation. However, several transporters mediating calcium import into mitochondria and chloroplasts have been identified. ...
Plant metabolism is constantly changing and requires input signals for efficient regulation. The mitochondrial calcium uniporter (MCU) couples organellar and cytoplasmic calcium oscillations leading to oxidative metabolism regulation in a vast array of species. In Arabidopsis thaliana, genetic deletion of AtMICU leads to altered mitochondrial calcium handling and ultrastructure. Here we aimed to further assess the consequences upon genetic deletion of AtMICU. Our results confirm that AtMICU safeguards intracellular calcium transport associated with carbohydrate, amino acid, and phytol metabolism modifications. The implications of such alterations are discussed.
... This notion is corroborated by our finding of elevated levels of MICU1 expression levels in TRPML1 knockout cells. Initially, MICU1 has been known to control mitochondrial calcium uptake and thereby safeguard against calcium overload and apoptosis (51). Concomitantly, MICU1 supports the structural integrity of cristae junctions in a calcium-dependent fashion (52), and it fine-tunes mitochondrial membrane potential and proper calcium handling (51). ...
... Initially, MICU1 has been known to control mitochondrial calcium uptake and thereby safeguard against calcium overload and apoptosis (51). Concomitantly, MICU1 supports the structural integrity of cristae junctions in a calcium-dependent fashion (52), and it fine-tunes mitochondrial membrane potential and proper calcium handling (51). In summary, we have described several mechanisms that may contribute to protect NK cells from calcium challenges and oxidative stress in mitochondria. ...
Cytotoxic lymphocytes eliminate cancer cells through the release of lytic granules, a specialized form of secretory lysosomes. This compartment is part of the pleomorphic endolysosomal system and is distinguished by its highly dynamic Ca2+ signaling machinery. Several transient receptor potential (TRP) calcium channels play essential roles in endolysosomal Ca2+ signaling and ensure the proper function of these organelles. In this study, we examined the role of TRPML1 (TRP cation channel, mucolipin subfamily, member 1) in regulating the homeostasis of secretory lysosomes and their cross-talk with mitochondria in human NK cells. We found that genetic deletion of TRPML1, which localizes to lysosomes in NK cells, led to mitochondrial fragmentation with evidence of collapsed mitochondrial cristae. Consequently, TRPML1−/− NK92 (NK92ML1−/−) displayed loss of mitochondrial membrane potential, increased reactive oxygen species stress, reduced ATP production, and compromised respiratory capacity. Using sensitive organelle-specific probes, we observed that mitochondria in NK92ML1−/− cells exhibited evidence of Ca2+ overload. Moreover, pharmacological activation of the TRPML1 channel in primary NK cells resulted in upregulation of LC3-II, whereas genetic deletion impeded autophagic flux and increased accumulation of dysfunctional mitochondria. Thus, TRPML1 impacts autophagy and clearance of damaged mitochondria. Taken together, these results suggest that an intimate interorganelle communication in NK cells is orchestrated by the lysosomal Ca2+ channel TRPML1.
... Recently, we reported that IP3-induced Ca 2+ rise in IMS activates MICU1 dimerization, leading to spatial opening of the cristae junction (CJ) 37 consequently decelerating the dynamics of the mitochondrial cristae membrane (CM) in MAM regions 38 (Fig. 4d). Therefore, we used structured illumination microscopy (SIM) to investigate the dynamics of CM in the whole mitochondria and the MAM regions of WT and AnxA5-KO cells. ...
... IMS Ca 2+ levels play a pivotal role in regulating the mitochondrial ultrastructure by triggering the process of MICU1 dimerization, which subsequently leads to the opening of CJ and ultimately causes mitochondrial fission 37,38,40 . Remarkably, our findings demonstrate that in AnxA5-KO cells, MICU1 dimerization and CJ openings are reduced, highlighting the role of AnxA5 in governing IMS Ca 2+ signaling and actively participating in the dynamic process of mitochondrial architecture modeling. ...
... Quantification of CM-kinetics in whole mitochondria and the MAM region was done as described elsewhere 37,38 . Briefly, WT and AnxA5-KO (HeLa) cells were transfected with ER-RFP and stained with Mitotracker Green/FM (MTG) and were recorded with live dual-color SIM imaging. ...
Annexin-A5 (AnxA5) is a Ca2+-dependent phospholipid-binding protein associated with the regulation of intracellular Ca2+ homeostasis. However, the precise role of AnxA5 in controlling mitochondrial Ca2+ signaling remained elusive. Here, we introduce a novel function of AnxA5 in regulating mitochondrial Ca2+ signaling. Our investigation revealed that AnxA5 orchestrates intermembrane space (IMS) Ca2+ access capacity upon high Ca2+ elevations induced by ER Ca2+ release, leading to AnxA5 translocation to the outer mitochondrial membrane. Through close association with the voltage-dependent anion channel (VDAC1), AnxA5 regulates the Ca2+-permeable state of VDAC1. By modulating IMS Ca2+ signaling, AnxA5 actively shapes mitochondrial ultrastructure and influences the dynamicity of the mitochondrial Ca2+ uniporter. Furthermore, our findings regarding AnxA5's localization in the VDAC1 microenvironment reveal its protective role in regulating cell death by controlling VDAC1's oligomeric state triggered by cisplatin-induced apoptosis. Our study uncovers AnxA5 as a regulator of VDAC1 in physiological and pathological conditions.
... Studies that have employed them indicate during resting conditions, MICU1 exists as a hexamer which stabilizes the CJ by interactions with OPA1 and MICOS (Gottschalk et al., 2019;Tomar et al., 2019; Figure 2A). Under resting conditions, MCU and EMRE subunits, located throughout the IMM, mediate Ca 2+ entry into the matrix (Gottschalk et al., 2019(Gottschalk et al., , 2022. With a physiological elevation of cytosolic Ca 2+ concentrations, the subsequent rise of IMS Ca 2+ levels triggers dissociation of the MICU1 hexamer resulting in opening of the CJ (Gottschalk et al., 2019(Gottschalk et al., , 2022 Figure 2B). ...
... Under resting conditions, MCU and EMRE subunits, located throughout the IMM, mediate Ca 2+ entry into the matrix (Gottschalk et al., 2019(Gottschalk et al., , 2022. With a physiological elevation of cytosolic Ca 2+ concentrations, the subsequent rise of IMS Ca 2+ levels triggers dissociation of the MICU1 hexamer resulting in opening of the CJ (Gottschalk et al., 2019(Gottschalk et al., , 2022 Figure 2B). MICU1 then forms homodimers or heterodimers with MICU2 that recruit MCU and EMRE subunits from the cristae to the IBM that regulate MCU cx activity (Gottschalk et al., 2019(Gottschalk et al., , 2022. ...
... With a physiological elevation of cytosolic Ca 2+ concentrations, the subsequent rise of IMS Ca 2+ levels triggers dissociation of the MICU1 hexamer resulting in opening of the CJ (Gottschalk et al., 2019(Gottschalk et al., , 2022 Figure 2B). MICU1 then forms homodimers or heterodimers with MICU2 that recruit MCU and EMRE subunits from the cristae to the IBM that regulate MCU cx activity (Gottschalk et al., 2019(Gottschalk et al., , 2022. ...
The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca ²⁺ ) uptake supports NVU function by buffering Ca ²⁺ and stimulating energy production. However, excessive mitochondrial Ca ²⁺ uptake causes toxic mitochondrial Ca ²⁺ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca ²⁺ uptake and efflux in the brain are mediated by the mitochondrial Ca ²⁺ uniporter complex (MCU cx ) and sodium/Ca ²⁺ /lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCU cx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca ²⁺ overloading. These findings suggest that combining MCU cx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCU cx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCU cx , or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.
... 52 MICU1's role in these functions is controlled by both Ca 2+ and PRMT1 that has been reported to catalyze the methylation of MICU1. 53,54 Thus, it is possible that binding of spermine to MICU1 might affect processes beyond the Ca 2+ flux through the mtCU. Although we did not find any spermine-induced alterations in the DJ m and used short exposure to the agonists in the Ca 2+ flux studies to minimize changes that might develop with a slower kinetics, future studies of spermine, SB202190, and kaempferol might also test directly the mitochondrial ultrastructure. ...
Mitochondrial Ca2+ homeostasis loses its control in many diseases and might provide therapeutic targets. Mitochondrial Ca2+ uptake is mediated by the uniporter channel (mtCU), formed by MCU and is regulated by the Ca2+-sensing gatekeeper, MICU1, which shows tissue-specific stoichiometry. An important gap in knowledge is the molecular mechanism of the mtCU activators and inhibitors. We report that all pharmacological activators of the mtCU (spermine, kaempferol, SB202190) act in a MICU1-dependent manner, likely by binding to MICU1 and preventing MICU1's gatekeeping activity. These agents also sensitized the mtCU to inhibition by Ru265 and enhanced the Mn2+-induced cytotoxicity as previously seen with MICU1 deletion. Thus, MCU gating by MICU1 is the target of mtCU agonists and is a barrier for inhibitors like RuRed/Ru360/Ru265. The varying MICU1:MCU ratios result in different outcomes for both mtCU agonists and antagonists in different tissues, which is relevant for both pre-clinical research and therapeutic efforts.
... In this regard, GDP was shown to displace FAs from UCP2 and to prevent proton shuttling [25]. In addition to its flippase-based uncoupling, UCP2 was shown to be indispensable for [2,5], which may also accomplish uncoupling via its action of mitochondrial Ca 2+ and cristae junction permeability [26,27]. ...
... Besides its impact on mitochondrial Ca 2+ uptake in cancer and aged cells [2,3], UCP2 profoundly impacts mitochondrial membrane potential in multiple ways. The function of UCP2 in the regulation of mitochondrial membrane potential crucially depends on the very recent discoveries of unequal dissemination of the potential across the IMM [26]. Importantly, MICU1, which controls the cristae junction (CJ) in a Ca 2+ -dependent manner serves as a crucial Ca 2+ -regulated discriminator for areas of high (i.e. ...
... cristae) and low (i.e. IBM) potential [26,27] (Fig. 2). Indeed, the CJ, however, is responsible for isolating the membrane potential generated by complexes I, III, and IV in the cristae [32,33] (Fig. 2, upper panel). ...
Mitochondrial uncoupling proteins UCP1 and UCP2 have a structural homology of app. 60%. They execute their mitochondria uncoupling function through different molecular mechanisms. Non-shivering thermogenesis by UCP1 is mediated through a transmembrane dissipation of the proton motive force to create heat during sympathetic stimulation. UCP2, on the other hand, modulates through the interaction with methylated MICU1 the permeability of the cristae junction, which acts as an isolator for the cristae-located mitochondrial membrane potential. In this mini-review, we discuss and compare the recently described molecular mechanism of UCP1 in brown adipose tissue and UCP2 in aged and cancer non-excitable cells that contribute to mitochondrial uncoupling, and the synergistic effects of both UCPs with the mitochondrial Ca2+ uptake machinery.
